Vascular permeability (VP) that mediate the exchange of plasma components between the vasculature and tissues, also contributes to angiogenesis and inflammation. A defect in endothelial-barrier results in 'vascular fragility'that often lead to irreversible VP, which is an underlying cause of many clinical conditions such as stroke, pulmonary edema, inflammation, atherosclerosis etc. Despite the fantastic progress in the VP research, the molecular mechanisms regulating endothelial-barrier function remains elusive. Protein kinase-B (Akt) is of great importance in the regulation of VP. This proposal is expected to elucidate how Akt utilizes its signaling partners in the differential regulation of endothelial-barrier in response to agents that augment or arrest VP. Our central hypothesis is that the cooperation between Akt and Src in regulating cytoskeletal and focal adhesion dynamics (via 2-Pix-Rac-GTPase signaling), AJ and TJ turnover (via GSK- 3/2-catenin, FoxO and ETS transcription factors), as well as initiation of a paracrine loop is involved in the coordination of various events in the endothelial-barrier function in response to VEGF (short-term Akt activation) and Ang-1 (long-term Akt activation). The rationale for the proposed research is that, once it is known mechanistically how Akt regulates VP, it is likely that endothelial-barrier function can be either up- or down-regulated therapeutically, which would be of singular importance in the management of a variety of clinical conditions. The combination of work proposed in this application is collectively expected to uncover how Akt modulates endothelial-barrier and VP in vivo in response to VEGF and Ang-1, which will have a positive impact because the identified components will likely provide new targets for preventive and therapeutic interventions. In addition, we expect our results to fundamentally advance the field of endothelial-barrier regulation and VP. Our specific aims are: (1) Identify the Akt signaling partners and determine the molecular mechanisms through which Akt regulates AJ and TJ protein turnover in response to VEGF and Ang-1. We hypothesize that the regulation of synthesis, interaction and turnover of proteins in adherens and tight-junctions by Akt and Src in response to VEGF and Ang-1 is mediated through GSK32- 2catenin pathway in association with FoxO and ETS. Once we identify GSK3-2catenin pathway as the 'molecular switch'in this differential regulation of VP by Akt, we will seek to confirm those findings in vivo. (2) Identify the Akt signaling partners and determine the molecular mechanisms through which Akt coordinates cytoskeletal and focal adhesion dynamics in response to VEGF and Ang-1. From our preliminary results, we postulate that the events leading to VP, such as expression and/or dynamics of integrins, focal adhesions, cytoskeletal proteins and cortical actin, are dependent on the ability of Akt to differentially modulate 2-Pix-Rac signaling. Once we identify these signaling partners and understand how Akt coordinates cytoskeletal and focal-adhesion dynamics in vitro, we will extend our findings in vivo. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because, once it is known mechanistically how Akt regulates endothelial-barrier function, it is likely that endothelial-barrier can be either up- or down-regulated therapeutically, which would be of singular importance in the management of a variety of clinical conditions. Identifying the molecular mechanisms through which Akt and its signaling partners regulate the sequence of events mediating endothelial-barrier function and vascular permeability represents a substantive departure from the status quo providing novel insights into the overall molecular mechanisms regulating the process. Because there are considerable similarities in molecular mechanisms regulating endothelial-barrier function in various tissues and clinical conditions, we expect that our findings will be relevant to the mission of NIH and be broadly interesting to researchers studying molecular mechanisms regulating endothelial barrier- function and vascular permeability in various physiological and pathological events such as pulmonary edema, stroke and a variety of ischemic and inflammation associated diseases.